Single concentration confirmation of uHTS hits from a small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay

Currently there are no small molecule tools to investigate the biological functions of apelin and its receptor. Apelin is the endogenous peptide ligand for the G-protein coupled receptor (GPCR) APJ (angiotensin II receptor-like 1, AGTRL-1 and APLNR). Until the discovery of apelin, APJ was an orphan GPCR. APJ is coupled to Gai, and has been shown in cell culture to inhibit adenylate cyclase. The more ..

Currently there are no small molecule tools to investigate the biological functions of apelin and its receptor. Apelin is the endogenous peptide ligand for the G-protein coupled receptor (GPCR) APJ (angiotensin II receptor-like 1, AGTRL-1 and APLNR). Until the discovery of apelin, APJ was an orphan GPCR. APJ is coupled to Gai, and has been shown in cell culture to inhibit adenylate cyclase. The APJ gene encodes a receptor that most closely resembles the angiotensin receptor AT1. However, the APJ receptor does not bind angiotensin II. Underscoring the emerging importance of the apelin/APJ system, recent studies have shown that apelin reduces the extent of atherosclerotic lesions in ApoE-/- mice, and opposes the development of abdominal aortic aneurysms. Additional research has revealed that APJ forms a heterodimer with the Ang II receptor AT1, and that this complex facilitates antagonism of Ang II signaling by apelin. Despite these exciting results, there remains a multitude of unanswered questions regarding the role of apelin and APJ in physiology and pathology. The project goal is to identify a chemical probe of apelin receptor function that transiently and reversibly activates the receptor. An antagonist or inhibitor receptor activation would provide a novel research tool to evaluate the role of apelin in cardiovascular and metabolic disease pathology.In this description we utilize enzyme-fragment complementation to directly measure GPCR activation. Unlike imaging or other second messenger assays, the DiscoveRx b-Arrestin assay allows for a direct measure of GPCR activation by detection of b-Arrestin binding to the APJ receptor. In this system, b-Arrestin is fused to an N-terminal deletion mutant of b-gal (termed the enzyme acceptor of EA) and the GPCR of interest is fused to a smaller (42 amino acids), weakly complementing fragment termed ProLink. In cells that stably express these fusion proteins, ligand stimulation results in the interaction of b-Arrestin and the Prolink-tagged GPCR, forcing the complementation of the two b-gal fragments and resulting in the formation of a functional enzyme that converts substrate to detectable signal. Antagonists would be expected to inhibit agonist activation of the receptor resulting in the inhibition of signal formation in this assay.

This assay is a follow-up to "uHTS identification of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay", AID 2521.

In this assay selected hits from the initial screen (AID 2521) were retested 4 times. The highest result is reported. Compounds with >50% activity are defined as "active" in this assay. To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below. Activity ScoringActivity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows: 1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 20 uM concentration:Scoring for the primary screen is not applicable in this assay.a. If outcome of the primary screen is inactive, then the assigned score is 0b. If outcome of the primary screen is inconclusive, then the assigned score is 10c. If outcome of the primary screen is active, then the assigned score is 20Scoring for Single concentration confirmation screening is applicable to this assay.d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay